Chromatin assembly and structure is highly regulated during DNA replication, gene expression, and progression through the cell cycle. Dysfunction of epigenetic factors, including methylation state, are associated with a variety of cancers. Histone lysine methyltransferases (HKMTs) are considered a new and important class of drug targets, however high throughput screening (HTS) assay formats are not available and reference inhibitors remain unknown. HTS assays and reference compounds would have significant potential to help elucidate function of these enzymes and to facilitate the development of anticancer agents. Reaction Biology Corporation (RBC) has developed extremely low cost reaction systems for many enzyme classes to serve markets for ultra high throughput screening (uHTS) drug discovery, large scale IC50 determinations and selectivity/toxicity profiling. The HotSpot platform employs a gold standard for ultralow volume radioisotope assays for kinase profiling. This service product for kinase profiling is widely accepted in the drug discovery community for kinase inhibitor development. Based on these skills, RBC seeks to develop a new assay platform using gold standard radioisotope assays for the large family of histone lysine methyltransferases (HKMTs). Through Phase I funding, the proposed research will focus on the following three aims: Aim 1 Develop a universal radioisotope-based gold standard for HKMTs with large signal/background ratio;Aim 2 Reduce assay costs by ultra-miniaturization and streamline assay protocols for robust HTS;Aim 3 Conduct HTS and identify new compounds for an HKMT using assays developed in Aims 1 and 2 by screening the libraries from RBC's chemical repository. This work will enable Phase II activities where RBC will expand its assay range across the large human HKMTs family and use this newly developed universal assay against large synthetic and natural compound libraries to discover potential molecules for research and therapeutic development. Selected lead compounds will be evaluated in cell based assays for anti-cancer activities PUBLIC HEALTH RELEVANCE: Chromatin assembly and structure is highly regulated during DNA replication, gene expression, and progression through the cell cycle. Dysfunction of epigenetic factors, including methylation state, are associated with a variety of cancers. Histone lysine methyltransferases are considered a new and important class of drug targets, however high throughput screening assay formats are not available and reference inhibitors remain unknown. HTS assays and reference compounds would have significant potential to help elucidate function of these enzymes and to facilitate the development of anticancer agents. Reaction Biology Corporation has developed extremely low cost reaction systems for many enzyme classes to serve markets for ultra high throughput screening drug discovery, large scale IC50 determinations and selectivity/toxicity profiling. The HotSpot platform employs a gold standard for ultralow volume radioisotope assays for kinase profiling. This service product for kinase profiling is widely accepted in the drug discovery community for kinase inhibitor development. Based on these skills, RBC seeks to develop a new assay platform using gold standard radioisotope assays for the large family of histone lysine methyltransferases.